Lawsonia inermis or (henna) is used as a cosmetic dye and medicinal plant for its strong antimycobacterial and other therapeutic effects. In this study, we investigated the phytochemical and antimycobacterial activity of L. inermis leaves methanol extract against Rifampicin sensitive and resistant mycobacterium tuberculosis (M.tb) strains. Fresh Sudanese henna leaves were collected from the University of Gezira campus garden, shade dried, ground and was extracted by methanol. Crude henna methanolic extract was then tested for antimycobacterial activity against eight M.tb strains sensitive and resistant to rifampicin, using drug susceptibility test. The phytoconstituents were identified by using gas chromatograph interfaced to a mass spectrometer (GC-MS). GC-MS analysis revealed the presence of seven phytochemical components, with prevailing of squalene (34.433%), Lawsone (20.185%) and Coumarin-4-carboxylic acid (23.328%). The crude methanolic extract of henna leaves demonstrated an inhibitory effect on all tested M.tb strains, with antimycobacterial activity being ≤ 50 mg/ml. Henna leaves crude methanolic extract is rich in phytoconstituents that may involve in its remarkable antimycobacterial activity against rifampicin sensitive and resistant M.tb strains.